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All Journal HAYATI Journal of Biosciences The Indonesian Biomedical Journal
ARTARINI, ANITA
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Earth & Planetary Sciences Public Health

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Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture Anindyajati; Afifah, Salma Aulia; Riani, Catur; Tan, Marselina Irasonia; Natalia, Dessy; Giri-Rachman, Ernawati Arifin; Artarini, Anita
HAYATI Journal of Biosciences Vol. 31 No. 3 (2024): May 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.3.572-580

Abstract

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period.
T118N Substitution of Hepatitis B X Protein Reduces Colony Formation of HepG2 Cells Artarini, Anita; Nurmalasari, Dewi Riskha; Permanasari, Silmi Citra; Riani, Catur; Tjandrawinata, Raymond Rubianto; Retnoningrum, Debbie Soefie
The Indonesian Biomedical Journal Vol 15, No 1 (2023)
Publisher : The Prodia Education and Research Institute (PERI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18585/inabj.v15i1.2095

Abstract

BACKGROUND: The acute Hepatitis B virus (HBV) infection usually ceases before six months, but chronic infection that lasts for more than six months might develop into liver cirrhosis and hepatocellular carcinoma (HCC). Viral particle load, HBV genotypes and association to the HBV x (HBx) gene mutations are the probable factors related to HCC occurrence. The mutation which leads to HBx T118N was found as the second most common HBx mutation in Indonesia, as compared to the known cancer-related HBx K130M/V131I mutant. However, the effect of T118N mutation and its combination with K130M/V131I on human hepatoma cells has not been elucidated well. Hence, this study was conducted to dissect the role of HBx T118N and its mutant combination in colony formation, as compared to the wild type HBx and cancer-related HBx K130M/V131I.METHODS: In this study, the genes encoding wild type HBx, HBx T118N, and HBx K130M/V131I mutations were obtained as synthetic gene. Meanwhile, the gene encoding HBx T118N/K130M/V131I mutations was successfully generated using site-directed mutagenesis. The optimum condition for colony formation assays was determined through Zeocin sensitivity test of HepG2 cells.RESULTS: Selection of HepG2 cells using Zeocin was determined at 200 µg/mL. Colony formation assays performed upon expression of HBx T118N and HBx T118N/K130M/V131I mutant proteins showed reduced colony numbers as compared to the expression of wild type HBx, similar to the effect from HBx K130M/V131I mutant expression.CONCLUSION: The HBx T118N and HBx T118N/K130M/V131I mutation caused less colony formation of HepG2 cells, similar to the K130/M131I mutation. This indicates a possible role of the T118N mutation in liver cancer development.KEYWORDS: colony formation assay, hepatitis B virus, HBx, T118N, K130M/V131I
Co-Authors Afifah, Salma Aulia Anindyajati CATUR RIANI DEBBIE SOEFIE RETNONINGRUM DESSY NATALIA Dewi Riskha Nurmalasari ERNAWATI ARIFIN GIRI-RACHMAN Permanasari, Silmi Citra Raymond R. Tjandrawinata Tan, Marselina Irasonia